BDNF Modulated KCC2 Ubiquitylation In Spinal Cord Dosal Horn Of Mice

Objective:K~+-Cl~-cotransporter 2(KCC2)is the major K~+-Cl~-co-transporter expressed in the dorsal horn of adult spinal cord.KCC2 operates to extrude Cl~-out of neurons and maintain the normal transmembrane Cl~-gradient,which is critical for inhibitory synaptic transmission.Previous studies have shown that peripheral nerve injuries downregulate KCC2 protein level in the spinal cord dorsal horn by activating Brain-Derived Neurotrophic Factor(BDNF)-Trk B signaling pathways,a crucial step that impairs spinal inhibition and evokes pathological pain.However,the molecular mechanisms for BDNF-Trk B signaling to downregulate KCC2 expression remain unclear.The current study was designed to investigate the role of Casitas B-lineage lymphoma b(Cbl-b),one of the E3 ubiquitin ligases,for BDNF to regulate KCC2 expression.Methods:Behavioral tests were used to examine the influence of intrathecal BDNF injection on the pain thresholds of mice.By performing biotinylation assays and co-immunoprecipitation,we studied the molecular interaction and protein ubiquitination.The pain model of spared nerve injury(SNI)was established to investigate the relationship between KCC2 ubiquitination and neuropathic pain.Results:(1)Intrathecal injection of BDNF(0.5μg)evoked mechanical allodynia,thermal hyperalgesia and cold allodynia.(2)Intrathecal injection of BDNF(0.5μg)significantly decreased the protein level of KCC2in spinal cord dorsal horn of intact mice and inhibited the KCC2 expression on the plasma membrane.(3)Intrathecal injection of BDNF(0.5μg)enhanced the ubiquitination level of KCC2.We observed that KCC2 was decorated by K48-linked ubiquitin rather than K63-linked ubiquitin after BDNF treatment.(4)We identified Cbl-b as the E3 ubiquitin ligase that ubiquitinated KCC2.Overexpression of constitutively active Cbl-b increased,whereas knockdown of Cbl-b decreased,the KCC2ubiquitination levels.(5)BDNF was able to activate Cbl-b in spinal cord dorsal horn.(6)Intrathecal injection of BDNF(0.5μg)promoted the tyrosine phosphorylation of KCC2and enhanced the interaction between KCC2 and Cbl-b.(7)sh RNA-mediated knockdown of spinal Cbl-b inhibited the mechanical allodynia,thermal hyperalgesia and cold allodynia evoked by BDNF.(8)Cbl-b-dependent KCC2 ubiquitination might contribute to the degradation of KCC2 in spinal dorsal horn after peripheral nerve injury.(9)Trk B blockade reversed KCC2 ubiquitination casued by nerve injury.Conclusion:BDNF activated Cbl-b to ubiquitinate and degradate KCC2 in spinal cord dorsal horn.